Bioluminescence is the emission of light from an organic molecule, such as xe2x80x9cluciferinxe2x80x9d, which has been oxidized by oxygen or one of its metabolites. The reaction is catalysed by a protein, usually known as a xe2x80x9cluciferasexe2x80x9d, or xe2x80x9cphotoproteinxe2x80x9d. The luciferases of beetles and fireflies utilize benzothiazole as a luciferin. For a given photoprotein additional substances, cofactors, may be required to generate light. These may include cations (such as magnesium), or cofactors such as NADH, FMN, or ATP; a fluor may also be included as an energy transfer acceptor. Reactions involving these cofactors, luciferases and luciferins result in the emission of light, which can occur in a spectrum of colors.
Photoproteins have been used in a variety of ex vivo and in vitro assays to determine levels of gene expression, presence and concentration of contaminants or energy levels (ATP). More recently, these enzymes have been used to monitor biological processes in living cells and animals. Generally, in luminescence assays, reaction substrates and other luminescence-activating reagents are introduced into a biological system suspected of expressing a reporter enzyme. Resultant luminescence, if any, is then measured using a photomuliplier tube, a charge coupled device. or any suitable radiant energy-measuring device in the form of a luminometer or camera. The assay can be rapid and sensitive, and may provide data quickly and easily, without the need for radioactive reagents, additional contrast reagents or dyes.
The most commonly used bioluminescent reporters emit in the blue to yellow-green range (250-560 nm). However, red light is transmitted through live tissue more efficiently than other wavelengths of visible light (see Cambell (1988), xe2x80x9cChemiluminescence: Principles and Applications in Biology and Medicinexe2x80x9d; and Jobis (1977)). By red-shifting the emission of bioluminescent reporters, it may be possible to enhance their utility for in vivo monitoring of biological processes, and provide additional reagents for multiparameter analysis by providing additional colors.
Multicolor functional assays have utility in analyses of coordinately regulated processes in ex vivo samples, cell lysates, living cells and living animals. Multiple colors of bioluminescent light from naturally occurring luminescent species have been identified, and in fact luciferase clones obtained from a single beetle species were shown to encode enzymes that emit at four different wavelengths (green to orange) (Wood et al. (1989) Science 244:700-702). With photoproteins that emit at different wavelengths, it may be possible to monitor multiple functions in vitro, in cells and in animals using spectrally resolved methods for analyses and imaging.
Luciferase genes are widely used as genetic reporters due to low background providing good signal to noise ratios, their non-radioactive nature, extreme sensitivity, broad dynamic range and linear response in various assays. (see, for example Welsh and Kay (1997) Curr Opin Biotechnol 8(5):617-22). Since as few as 10xe2x88x9220 moles of the firefly luciferase can be detected, luciferase assays for gene activity are used in virtually every experimental biological system, including prokaryotic and eukaryotic cell cultures, transgenic plants, cells and animals, and cell-free expression systems. Similarly, luciferase assays of ATP are highly sensitive, enabling detection to below 10xe2x88x9216 moles.
Currently, luciferase genes from a wide variety of vastly different species, particularly the luciferase genes of Photinus pyralis (the common firefly of North America), Pyrophorus plagiophathalamus (the Jamaican click beetle), Renilla reniformis (the sea pansy), and several bacteria (e.g., Photorhabdus luminescens and Vibrio spp), are used as luminescence reporter genes. Amino acid substitutions in the active sites of luciferase clones have been reported to alter wavelength of emission (Kajiyama et al. (1991) Prot. Eng. 4:691). However, red-emitting clones of a well-characterized luciferase with readily available sources of substrate have not been reported.
Relevant Literature
Engineering of luciferases has been reported. Sung and Hang (1998) Photochem Photobiol 68(5):749-53 disclose that the N-terminal amino acid sequences of the firefly luciferase are important for the stability of the enzyme. Branchini et al. (1998) Biochemistry 37(44):15311-9 performed site-directed mutagenesis of histidine 245 in firefly luciferase. White et al. (1996) Biochem J 319 (Pt 2):343-50. showed an improvement in the thermostability of the North American firefly luciferase by saturation mutagenesis at position 354. Kajiyama and Nakano (1994) Biosci Biotechnol Biochem 58(6):1170-1 constructed firefly luciferase mutants from Luciola lateralis in which Ala at position 217 was replaced by each of three hydrophobic amino acid residues (lie, Leu,. and Val). U.S. Pat. No. 5,401,629 Harpold, et al. describes assay methods and compositions useful for measuring the transduction of an intracellular signal.
Contag et al. (1997) Photochem Photobiol 66(4):523-31 describes the use of bioluminescence to monitor gene expression in living mammals. Viral promoters fused to firefly luciferase as transgenes in mice allowed external monitoring of gene expression both superficially and in deep tissues. In vivo bioluminescence was detectable using either intensified or cooled charge-coupled device cameras, and could be detected following both topical and systemic delivery of substrate. U.S. Pat. No. 5,650,135, Contag, et al., entitled xe2x80x9cNon-invasive localization of a light-emitting conjugate in a mammalxe2x80x9d also describes the use of in vivo bioluminescence.
Contag et al. (1995) Mol Microbiol 18(4):593-603 describe a method for detecting bacterial pathogens in a living host; used to evaluate disease processes for strains of Salmonella typhimurlum that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive.
Hooper et al. (1990) J Biolumin Chemilumin 5(2):123-30 review low-light-level imaging, with particular reference to charge-coupled device (CCD) cameras. Detectors for sensitive imaging are described and compared, including various CCDs and photon-counting devices. Image analysis techniques based on digital image processing may be applied to quantify luminescent processes with these detectors. Images of luciferase gene expression in single mammalian cells have been obtained using a particular high-sensitivity intensified CCD camera.
The nucleotide sequence of the optimized firefly luciferase gene present in the plasmid, pGL3, may be accessed at Genbank (no. U47298), and for convenience is provided in the SEQLIST as SEQ ID NOs:7 and 8. This sequence is based on the wild-type Photinus pyralis sequence, accessible as Genbank no. M15077, or in the SEQLIST as SEQ ID NOs:11 and 12. The sequence of the luciferase from Luciola lateralis is accessible as Genbank no. U51019, or in the SEQLIST as SEQ ID NOs:9 and 10.
Nucleic acid sequences, and predicted amino acid sequences, for mutated forms of the firefly luciferase gene are provided. Gene products from these mutated forms are characterized by an altered light emission, where the normal, or wild-type, yellow-green peak at 560 nm is shifted to a orange-red peak at 610 nm, hence a red-shift. The nucleic acid compositions and resulting enzymes find use in various systems as a reporter gene, and are of particular interest for use as a reporter in multicolor assays and with in vivo systems, because of the relative increase in tissue penetration by red light over shorter wavelengths. The luciferase mutants may be used in a range of biological investigations, including the detection, location and measurement of microbes (protozoa, bacteria, viruses); detection and location of cancer cells; measurement of enzymes, intracellular signaling and other turnover reactions in cells or fluids; DNA and RNA binding assays; immunoassays and other protein assays. The red-shifted luciferase may be combined in such assays with luciferases emitting at other wavelengths, in order to monitor multiple processes simultaneously.